Lipid membrane, method for measuring membrane permeability, and method for screening

ABSTRACT

The object of the present invention is to provide a lipid membrane whose permeability to substances is high and strongly correlated with the permeability of biomembranes to drugs and which therefore is suitable for rapid measurement. The present invention provides a membrane comprising an unsaturated C 7 -C 9  hydrocarbon and a lipid.

This application is a 371 of PCT/JP01/02346, filed Mar. 23, 2001

TECHNICAL FIELD

The present invention relates to lipid membranes, more specificallylipid membranes that can be used for measuring the membrane permeabilityof substances.

BACKGROUND ART

Lipid membranes have heretofore been used as detection membranes or thelike in sensors or measuring devices. For example, they have been usedfor the detection or assay of various substances such as metal ions,cyanide ions, alcohols and enzymes.

Lipid membranes have also been used for evaluating the permeability ofbiomembranes which mediate various reactions in the body, e.g.,substance and/or energy transfer, metabolism, signal transduction, etc.

For example, the permeability of drugs across lipid membranes is closelyrelated to gastrointestinal absorption and in vivo trans-tissue deliveryof oral formulations, so it is a critical property in developingpharmaceuticals. In addition to drugs, it is also critical to evaluatethe membrane permeability of substances which adversely affect livingorganisms (e.g., toxic substances, carcinogenic substances, etc.).

To determine the membrane permeability of substances in the body by anin vitro technique, there is a need to use a membrane whose permeabilityto substances is strongly correlated with the in vivo permeability ofbiomembranes to substances. In addition to this, throughput speed andcost are critical requirements for industrial application of suchmembranes. For example, when such a membrane is used to evaluate themembrane permeability of drugs in pharmaceutical development, themembrane is required to provide high throughput speed because a numerousnumber of compounds are required to be screened.

For in vitro determination of the membrane permeability of substances,several techniques are conventionally used, such as a technique usingisolated organs, a technique using intestinal epithelium-derived cells,etc. However, because of their low throughput speed, these techniquesare unable to provide rapid screening of a large number of substances.

Other techniques are also known for this purpose, e.g., an artificialmembrane permeation assay using an artificial lipid membrane formed on a96-well plate (see Manfred KANSY, Frank SENNER, Klaus GUBERNATOR;Journal of Medicinal Chemistry 1998, 41, 1007-1010). A lipid membraneprepared from a lipid(s) and an organic solvent(s) is used as anartificial lipid membrane in this technique. Major features of thistechnique include, for example, the ability to perform a parallel assayof many substances at the same time and low running cost due to the needto use only a small amount of substances for assay. However, themembrane used in the disclosed technique is disadvantageous in that itnot only has a weak correlation with the in vivo permeability ofbiomembranes to substances, but it also involves difficulty inevaluating low-permeability substances and requires a long time forevaluation because the membrane is less permeable to substances.

Membranes used for measuring the membrane permeability of substances areknown from, for example, Ken-ichi INUI, Katsue TABARA, Ryohei HIRI,Akemi KANEDA, Shozo MURANISHI, Hitoshi SEZAKI; Journal of Pharmacy andPharmacology, 1977, 29, 22-26, which discloses a membrane that isprepared from living organism (rat) derived-membrane componentsdissolved in n-decane. This membrane is characterized by having a strongcorrelation with the permeability of biomembranes to substances, but itis still disadvantageous in that it involves difficulty in evaluatinglow-permeability substances and requires a long time for evaluationbecause the membrane is less permeable to substances.

Also, a membrane prepared from dodecane, phosphatidylcholine and1,9-decadiene is known from Manfred KANSY, Frank SENNER, KlausGUBERNATOR; Journal of Medicinal Chemistry 1998, 41, 1007-1010. However,such a membrane is similarly disadvantageous in that it requires a longtime for measurement and involves difficulty in distinguishing betweenlow-permeability substances because the membrane is less permeable tosubstances and a further disadvantage is in that it has a weakcorrelation with the permeability of biomembranes to substances.

DISCLOSURE OF THE INVENTION

As discussed above, conventional membranes used for measuring themembrane permeability of substances are disadvantageous in that theyrequire a long time for measurement and involve difficulty indistinguishing between low-permeability substances because thesemembranes are less permeable to substances and that they have a weakcorrelation with the permeability of biomembranes to substances. Theobject of the present invention is to overcome these disadvantages andprovide a lipid membrane whose permeability to substances is high andstrongly correlated with the permeability of biomembranes to substancesand which therefore is suitable for rapid measurement.

As a result of our careful studies, it has been found that the use ofunsaturated C₇-C₉ hydrocarbons was effective in preparing ahigh-permeability lipid membrane suitable for rapid measurement, therebyfinally completing one aspect of the invention.

We have further found that the use of unsaturated C₇-C₉ hydrocarbons andmaterials negatively charged at around neutral pH and/or lipidsnegatively charged at around neutral pH was effective in preparing alipid membrane whose permeability to drugs was high and stronglycorrelated with the in vivo permeability of biomembranes to drugs andwhich therefore was suitable for rapid measurement; in this way, anotheraspect of the invention was finally completed.

Namely, the present invention provides a lipid membrane comprising anunsaturated C₇-C₉ hydrocarbon and a lipid.

Preferably, the lipid membrane further comprises a material negativelycharged at around neutral pH. Alternatively, the lipid preferably isnegatively charged at around neutral pH.

The unsaturated C₇-C₉ hydrocarbon preferably is heptadiene, octadiene ornonadiene, more preferably 1,6-heptadiene, 1,7-octadiene or1,8-nonadiene.

The lipid membrane of the present invention may comprise one or moreadditional components, such as a carrier for transporting a specificsubstance (i.e., specific transporter).

The present invention also provides a method for measuring membranepermeability, which comprises the step of measuring the membranepermeability of a substance using the above-mentioned lipid membrane.

Further, the present invention provides a kit for measuring membranepermeability, which comprises an unsaturated C₇-C₉ hydrocarbon and alipid. Such a kit may further comprise a support (e.g., a filter paper)capable of holding a lipid membrane prepared from an unsaturated C₇-C₉hydrocarbon and a lipid.

The present invention provides a screening method, which comprises thesteps of:

measuring the membrane permeability of a substance using theabove-mentioned lipid membrane; and

selecting the substance if it has the desired membrane permeability.

Further, the present invention provides a screening kit comprising anunsaturated C₇-C₉ hydrocarbon, a lipid and an instruction manual foruse.

The measuring method of the present invention may also be combined witha method for determining membrane's active transport. A screening methodcomprising such combined methods and a screening kit used therefor arealso included within the scope of the present invention.

The lipid membrane of the present invention not only evaluates thepermeability of substances across biomembranes, but it can also beapplied to sensors or measuring devices for chemical substances or thelike.

BEST MODE FOR CARRYING OUT THE INVENTION

This application claims the priority of Japanese Patent Application Nos.2000-82177 and 2000-184973, the disclosures of which are herebyincorporated by reference in their entirety.

The unsaturated C₇-C₉ hydrocarbon used herein may be linear or branched,examples of which include heptadiene, octadiene and nonadiene.

Examples of heptadiene include (Z)-1,3-heptadiene, (Z)-1,4-heptadiene,(Z)-1,5-heptadiene, 1,6-heptadiene, (E)-1,3-heptadiene,(E)-1,4-heptadiene, (E)-1,5-heptadiene, (2Z,4Z)-2,4-heptadiene,(2Z,5Z)-2,5-heptadiene, (2Z,4E)-2,4-heptadiene, (2Z,5E)-2,5-heptadiene,(2E,4Z)-2,4-heptadiene, (2E,5Z)-2,5-heptadiene, (2E,4E)-2,4-heptadiene,(2E,5E)-2,5-heptadiene, (3Z,5Z)-3,5-heptadiene, (3Z,5E)-3,5-heptadiene,(3E,5Z)-3,5-heptadiene and (3E,5E)-3,5-heptadiene, with 1,6-heptadienebeing preferred.

Examples of octadiene include (Z)-1,3-octadiene, (Z)-1,4-octadiene,(Z)-1,5-octadiene, (Z)-1,6-octadiene, (E)-1,3-octadiene,(E)-1,4-octadiene, (E)-1,5-octadiene, (E)-1,6-octadiene, 1,7-octadiene,(2Z,4Z)-2,4-octadiene, (2Z,5Z)-2,5-octadiene, (2Z,6Z)-2,6-octadiene,(2Z,4E)-2,4-octadiene, (2Z,5E)-2,5-octadiene, (2Z,6E)-2,6-octadiene,(2E,4Z)-2,4-octadiene, (2E,5Z)-2,5-octadiene, (2E,6Z)-2,6-octadiene,(2E,4E)-2,4-octadiene, (2E,5E)-2,5-octadiene, (2E,6E)-2,6-octadiene,(3Z,5Z)-3,5-octadiene, (3Z,5E)-3,5-octadiene, (3E,5Z)-3,5-octadiene and(3E,5E)-3,5-octadiene, with 1,7-octadiene being preferred.

Examples of nonadiene include (Z)-1,3-nonadiene, (Z)-1,4-nonadiene,(Z)-1,5-nonadiene, (Z)-1,6-nonadiene, (Z)-1,7-nonadiene,(E)-1,3-nonadiene, (E)-1,4-nonadiene, (E)-1,5-nonadiene,(E)-1,6-nonadiene, (E)-1,7-nonadiene, 1,8-nonadiene,(2Z,4Z)-2,4-nonadiene, (2Z,5Z)-2,5-nonadiene, (2Z,6Z)-2,6-nonadiene,(2Z,7Z)-2,7-nonadiene, (2Z,4E)-2,4-nonadiene, (2Z,5E)-2,5-nonadiene,(2Z,6E)-2,6-nonadiene, (2Z,7E)-2,7-nonadiene, (2E,4Z)-2,4-nonadiene,(2E,5Z)-2,5-nonadiene, (2E,6Z)-2,6-nonadiene, (2E,7Z)-2,7-nonadiene,(2E,4E)-2,4-nonadiene, (2E,5E)-2,5-nonadiene, (2E,6E)-2,6-nonadiene,(2E,7E)-2,7-nonadiene, (3Z,5Z)-3,5-nonadiene, (3Z,6Z)-3,6-nonadiene,(3Z,5E)-3,5-nonadiene, (3Z,6E)-3,6-nonadiene, (3E,5Z)-3,5-nonadiene,(3E,6Z)-3,6-nonadiene, (3E,5E)-3,5-nonadiene and (3E,6E)-3,6-nonadiene,with 1,8-nonadiene being preferred.

These heptadiene, octadiene and nonadiene compounds may be used alone orin combination as an unsaturated C₇-C₉ hydrocarbon component. The lipidmembrane of the present invention may comprise, as an organic solventcomponent, an unsaturated C₇-C₉ hydrocarbon either alone or incombination with other organic solvents.

Examples of the lipid used herein include saturated fatty acids,unsaturated fatty acids, phospholipids and cholesterols.

Saturated fatty acids include, but are not limited to, lauric acid,myristic acid, palmitic acid, stearic acid, arachidic acid and behenicacid.

Unsaturated fatty acids include, but are not limited to, palmitoleicacid, linolenic acid, linoleic acid, oleic acid and arachidonic acid.

Phospholipids include, but are not limited to, phosphatidylcholine,phosphatidylglycerol, phosphatidyl-inositol, phosphatidylserine,phosphatidylethanolamine and sphingomyelin.

These lipids may be used alone or in combination.

Examples of the material negatively charged at around neutral pH usedherein include stearic acid, phosphatidylserine andphosphatidylinositol. These materials may be used alone or incombination.

The lipid membrane of the present invention preferably comprises thelipid and the unsaturated C₇-C₉ hydrocarbon at a ratio (i.e.,lipid/unsaturated C₇-C₉ hydrocarbon ratio by weight) of 0.1% to 20%,more preferably 1% to 10%, and most preferably 1% to 2%.

In the case where the lipid membrane of the present invention comprisesa material negatively charged at around neutral pH, the membranepreferably comprises the material in an amount of about 0.2 mmol/L toabout 50 mmol/L, more preferably about 2 mmol/L to about 25 mmol/L,based on the sum of the lipid membrane components.

As used herein, the term “around neutral pH” generally refers to a rangeof pH 5.0 to pH 9.0, preferably pH 5.5 to pH 8.0, more preferably pH 6.0to pH 7.5.

In addition, the term “negatively charged at around neutral pH” meansthat the material and/or lipid carries a negative charge in a givensolvent at around neutral pH.

The lipid membrane of the present invention may further comprise one ormore additional components other than those stated above. Suchadditional components may be selected as appropriate for the nature ofsubstances, living organisms or tissues to be evaluated, etc. Forexample, the lipid membrane may comprise a carrier for transporting aspecific substance (specific transporter).

The thickness of the lipid membrane of the present invention may beselected as appropriate for the nature of substances, living organismsor tissues to be evaluated, etc.

The lipid membrane of the present invention may be formed on a certainsupport. A preferred support is a porous sheet or film, such as filterpaper. The support is preferably made of a hydrophobic material such asPTFE (polytetrafluoroethylene) or hydrophobic PVDF (polyvinylidenedifluoride), more preferably hydrophobic PVDF. It is desirable to use asupport with a pore size of 0.01 to 20 μm, preferably 0.05 to 10 μm,more preferably 0.1 to 5 μm, and most preferably around 0.1 to 1 μm.

Alternatively, the lipid membrane of the present invention may be formedso as to cover small pore openings having a diameter of about 0.5 to 2mm, preferably around 1 mm.

The lipid membrane of the present invention can be prepared by mixingthe above unsaturated C₇-C₉ hydrocarbon and the above lipid in astandard manner and then treated as described in Manfred KANSY, FrankSENNER, Klaus GUBERNATOR; Journal of Medicinal Chemistry 1998, 41,1007-1010 or elsewhere.

The measuring method of the present invention comprises the step ofmeasuring the membrane permeability of substances using the lipidmembrane of the present invention. In this method, the lipid membrane tobe used for measurement may be formed and stored prior to themeasurement or it may be formed immediately before the measurement. Thelipid membrane is preferably formed on a support, more preferably on ahydrophobic support. A solution containing a test substance is injectedinto one side (called “side A”) of the resulting lipid membrane, while asolvent free of the test substance is injected into the other side(called “side B”). The solvent injected into side B is preferablyidentical with a solvent for the solution of the test substance injectedinto side A. After a given time period has passed, the amount of thetest substance that has permeated across the lipid membrane from side Ato side B is determined. Instead of direct measurement of the testsubstance that has permeated across the lipid membrane, the amount ofthe test substance remaining on side A (i.e., without permeating acrossthe lipid membrane) may also be measured to calculate the amount of thetest substance that has permeated across the lipid membrane.

The lipid membrane may be placed in any direction with respect to thegravity vector, for example, in a direction either parallel orperpendicular to the gravity vector. Also, the direction of substancepermeation is not particularly limited. For example, the substancepermeation may be set in a direction parallel, opposite or perpendicularto the gravity vector.

The measurement may be performed using a container divided by the lipidmembrane into two compartments or using two separate containers, one ofwhich is provided for a solution of the test substance and the other isprovided for a solvent free of the test substance. For example, acombination of an upper cylindrical container having a filter at thebottom and a lower container having an open top can be used formeasurement purposes. Preferably, the lower end of the upper containerand the upper end of the lower container are of the same size. The lowercontainer is filled with the solvent free of the test substance and theupper container is placed on the lower container. After the lipidmembrane of the present invention is formed on the filter, the solutionof the test substance is injected into the upper container and theamount of the test substance in the lower container is determined aftera given time period has passed. The upper container is placed on thelower container such that the filter of the upper container contacts thesolvent in the lower container. The upper and lower containers should beso fixed as to prevent fluid leakage from between the mating surfaces asthey hold the filter. Rubber packing or the like may be used for thispurpose. The filter is not always required to be attached to the bottomof the upper container.

In the case where rapid measurement of membrane permeability isperformed on two or more test substances, it is desirable to use amulti-well plate, which may be used as a lower container. In this case,each well of the multi-well plate is filled with the solvent free of thetest substance and then covered with a porous film such as filter paper.After an upper plate perforated with holes at positions corresponding toindividual wells (used as an upper container) is placed on themulti-well plate, a lipid membrane is formed on the porous film withineach hole, into which the solution of the test substance is theninjected. Alternatively, the upper plate may have a filter at the bottomof each hole. For example, a chamber plate for chemotaxis assay or thelike may be used.

In either of these cases, the solvent free of the test substance may beinjected into the upper container, while the solution of the testsubstance may be injected into the lower container.

The amount of the substance that has permeated across the lipid membranemay be determined using, for example, absorbance measurement, HPLC, TLC(thin-layer chromatography), GC-MS (gas chromatography/massspectrometry), LC-MS (liquid chromatography/mass spectrometry),fluorescence analysis, NMR, IR and CE (capillary electrophoresis),preferably absorbance measurement, HPLC and LC-MS.

In the present invention, as shown in the examples below, the membranepermeability of substances was measured as described in Manfred KANSY,Frank SENNER, Klaus GUBERNATOR; Journal of Medicinal Chemistry 1998, 41,1007-1010. In addition to this, the measurement may be performed asdescribed in, for example, Michael THOMPSON, R. Bruce LENNOX, R. A.MCCLELLAND, Analytical Chemistry, 1982, 54, 76-81 or Tian-xiang XIANG,Bradley D. ANDERSON, Journal of Pharmaceuitical Science, 1994, 83, 1511.

To perform screening of substances using the lipid membrane of thepresent invention, the membrane permeability of test substances ismeasured as stated above and test substances with the desired membranepermeability are then selected. In this screening, a certain referencesubstance may be used to select substances with higher or lower membranepermeability than the reference substance. Alternatively, testsubstances with the same membrane permeability as the referencesubstance may be selected.

The screening kit of the present invention, which is helpful ineffecting such screening rapidly and conveniently, comprises anunsaturated C₇-C₉ hydrocarbon, a lipid and an instruction manual foruse. In addition to these components, the kit may further comprise asupport for the lipid membrane (e.g., filter paper, preferably ahydrophobic filter paper), a multi-well plate for use in measurement ofmembrane permeability, a solvent, a reference substance for evaluationof membrane permeability, a carrier for transporting a specificsubstance (specific transporter), a solvent for dissolving testsubstances, etc. Alternatively, the screening kit of the presentinvention may comprise a pre-formed lipid membrane instead of anunsaturated C₇-C₉ hydrocarbon and a lipid. Such a preformed lipidmembrane may be on a support.

The present invention will be further described in the followingexamples. The examples are provided for illustrative purposes only, andare not intended to limit the scope of the invention. For example,various conditions including the thickness of a lipid membrane to beprepared, the concentration of lipid and the pH of a buffer can beadjusted as appropriate for the nature of substances, living organismsor tissues to be evaluated, etc.

EXAMPLES

Preparation of Lipid Membranes

Each well (volume: 360 μL) of a 96-well plate (flat-bottomed plate 3072,Falcon) was filled with a 50 mmol/L sodium phosphate buffer containing5% dimethyl sulfoxide (pH 6.5 to pH 7.0; hereinafter referred to as“buffer”) and the 96-well plate was then covered with a filter plate(Millipore Multiscreen filter plate, hydrophobic PVDF MAIPN4510;thickness: 0.45 μm). At that time, it was confirmed that there was noair bubble between the filter plate and the buffer in the 96-well plate.Each of the components shown in Table 1 (membrane composition) wasdissolved in 1,7-octadiene in the amount also indicated in Table 1. Theresulting 1,7-octadiene solution was added to the filter within eachwell in a volume of 4 to 5 μL per well to form a membrane on the filter(Examples 1 and 2).

Similarly, a solution containing phosphatidylcholine and 1,6-heptadiene(2:98) was added to the filter within each well in a volume of 4 to 5 μLper well to form a membrane on the filter (Example 3). A solutioncontaining phosphatidylcholine and 1,7-octadiene (2:98) was added to thefilter within each well in a volume of 4 to 5 μL per well to form amembrane on the filter (Example 4). A solution containingphosphatidylcholine and 1,8-nonadiene (2:98) was added to the filterwithin each well in a volume of 4 to 5 μL per well to form a membrane onthe filter (Example 5).

In control experiments, a solution containing phosphatidylcholine,cholesterol and 1,9-decadiene (2:1:97) was prepared and added to thefilter within each well in a volume of 4 to 5 μL per well to form amembrane on the filter (Comparative Example 1). A solution containingphosphatidylcholine and 1,9-decadiene (2:98) was added to the filterwithin each well in a volume of 4 to 5 μL per well to form a membrane onthe filter (Comparative Example 2).

TABLE 1 Phosphatidyl- Phosphatidyl- Phosphatidyl- Phosphatidyl- Steariccholine ethanolamine serine inositol Cholesterol acid (%, w/w) (%, w/w)(%, w/w) (%, w/w) (%, w/w) (%, w/w) Example 1 2 0 0 0 1 0.2 Example 20.75 0.75 0.25 0.25 1 0Measurement of Membrane Permeability

Each of the compounds shown in Tables 2 and 3 was dissolved in thebuffer to give a 0.5 mmol/L solution, which was then used as a samplesolution in a volume of 100 to 200 μL (volume of the sample solutionadded to each well: V_(dn)). The sample solutions thus prepared wereadded to individual wells where the membrane had been formed as statedabove, followed by allowing them to stand for 2 to 15 hours (permeationtime: t) with the plate covered with a lid. After the filter plate wasremoved, an aliquot (200 μL) was taken from each well of the lower96-well plate and used as a test solution.

The resulting test solutions (200 μL each) were assayed for theirabsorbance (OD_(ac)) over the wavelength range from 250 to 450 nm atintervals of 10 to 20 nm. As a reference solution, the sample solutionused in Example 1 or 3 was used on its own (undiluted referencesolution) or diluted 4.8-fold (V/V) in the buffer (diluted referencesolution). Namely, either the undiluted or the diluted referencesolution was used in a volume of 200 μL and similarly assayed for itsabsorbance (OD_(ref)). The permeability coefficient was calculatedaccording to the following equation:Permeability coefficient (P)=−2.30333 (V _(dn) ×V _(ac)/(V _(dn) ×V_(ac)))/(S×t)×log(1−flux %/100)whereflux %=OD _(ac) /OD _(ref) ×A×100

OD_(ac): measured absorbance of the test solution

OD_(ref): measured absorbance of the reference solution

V_(ac): volume of each well in the lower plate (360 μL)

V_(dn): volume of the sample solution added to each well (100 to 200 μL)

A: V_(dn)×V_(ac) (undiluted reference solution) or 1 (diluted referencesolution)

S: area of the membrane (0.266 cm²)

t: permeation time (sec).

Tables 2 and 3 show the calculated permeability coefficients (P) ofindividual compounds.

With respect to the membranes of Examples 1 and 2 and ComparativeExample 1, the correlation coefficient (R) was further calculated usingthe following approximation curve obtained from human intestinalabsorption (Fa) data of individual compounds, which were found in thefollowing References *1 to *4:Approximation curve:Fa (%)=(1−exp(−a×P))×100where

exp: exponential

a: coefficient.

References:

-   *1 Matthew D. Wessel et al., J. Chem. Inf. Comput. Sci., 38, 1998,    726-735-   *2 Mehran Yazdanian et al., Pharm. Res., Vol. 15, No. 9, 1998,    1490-1494-   *3 Gerald K. McEvoy, AHFS, 1998-   *4 Manfred Kansy et al., J. Med. Chem., Vol. 47, No. 7, 1998,    1007-1010

Table 2 also shows the calculated correlation coefficients (R) and humanintestinal absorption (Fa) data of individual compounds.

TABLE 2 Compar- ative Human Example 1 Example 2 Example 1 intestinal (P)(P) (P) absorption Compound pH 7.0 pH 6.5 pH 6.5 (Fa, %) Acebutolol4.45E-06 3.67E-06 1.91E-07 90*¹ Acetaminophen 8.17E-06 1.79E-06 4.98E-0880*¹ Aciclovir 7.97E-08 8.89E-08 3.87E-08 20*² Amiloride 2.69E-066.73E-07 6.10E-08 50*³ Atenolol 8.02E-07 8.64E-07 4.18E-07 50*¹Ceftriaxone 1.37E-06 2.25E-07 1.66E-08  1*⁴ Cefuroxime 1.60E-07 4.43E-082.76E-08  5*¹ Chlorothiazide 2.24E-06 2.71E-07 4.98E-08 13*¹ Cytarabine8.89E-07 3.87E-08 3.87E-08 20*³ Doxycycline 7.07E-06 2.35E-05 1.52E-0695*³ Enalapril 8.02E-07 1.37E-06 1.09E-06 65*³ Furosemide 3.09E-069.02E-07 1.34E-07 61*¹ Guanabenz 1.95E-05 1.04E-05 4.14E-06 75*¹Hydrochlorothiazide 1.34E-06 1.55E-06 8.64E-07 67*¹ Hydrocortisone8.84E-06 1.44E-05 3.18E-06 91*¹ Metoprolol 9.42E-06 6.88E-06 7.49E-0795*¹ Nadolol 3.28E-06 1.14E-06 5.26E-07 35*¹ Naltrexone 1.12E-054.50E-06 9.61E-06 96*³ Oxytetracycline 3.31E-06 5.87E-06 8.31E-07 60*³Pindolol 1.10E-05 7.94E-06 2.71E-08 90*¹ Practolol 1.01E-06 1.53E-064.36E-07 100*¹  Pravastatin 2.69E-06 6.12E-07 7.21E-08 34*¹ Procainamide4.58E-06 3.89E-06 1.17E-07 85*³ Quinidine 2.56E-05 1.47E-05 4.51E-0680*¹ Ranitidine 1.22E-06 2.19E-06 1.34E-07 50*¹ Sulfasalazine 3.24E-061.07E-06 3.12E-07 65*¹ Sulpiride 1.34E-06 2.23E-06 8.33E-08 35*⁴Tetracycline 2.65E-06 7.62E-06 1.40E-06   77.5*³ Timolol 9.13E-061.19E-05 1.05E-06 90*¹ Correlation coefficient 0.798 0.847 0.534 — (R)between P and hu- man intestinal absorption

TABLE 3 Comparative Example 3 Example 4 Example 5 Example 2 Compound (P)(P) (P) (P) Hydrocortisone 4.36E-05 1.87E-05 1.29E-05 1.75E-06Propranolol 4.38E-05 2.15E-05 2.46E-05 1.25E-05 Ketoprofen 5.05E-051.36E-05 1.68E-05 9.22E-06 Procainamide 1.99E-06 2.42E-07 4.51E-073.14E-07 Furosemide 4.97E-06 1.66E-06 3.12E-06 2.44E-07Hydrochlorothiazide 5.86E-06 1.52E-06 1.82E-06 7.51E-07

INDUSTRIAL APPLICABILITY

The present invention provides lipid membranes whose permeability tosubstances is high and strongly correlated with the permeability ofbiomembranes to substances and which therefore is suitable for rapidmeasurement. Such lipid membranes have great utility.

1. A lipid membrane comprising an unsaturated C₇-C₉ hydrocarbon and alipid.
 2. The lipid membrane according to claim 1, which furthercomprises a material negatively charged at around neutral pH.
 3. Thelipid membrane according to claim 2 wherein the unsaturated C₇-C₉hydrocarbon is heptadine, octadine or nonadiene.
 4. The lipid membraneaccording to claim 2 wherein the unsaturated C₇-C₉ hydrocarbon is1,6-heptadiene, 1,7-octadiene or 1,8-nonadiene.
 5. The lipid membraneaccording to claim 2, wherein the unsaturated C₇-C₉ hydrocarbon isheptadiene, octadiene, or nonadiene.
 6. The lipid membrane according toclaim 2, wherein the unsaturated C₇-C₉ hydrocarbon is 1,6-heptadiene,1,7-octadiene, or 1,8-nonadiene.
 7. The lipid membrane according toclaim 1, wherein the lipid is negatively charged at around neutral pH.8. The lipid membrane according to claim 7 wherein the unsaturated C₇-C₉hydrocarbon is heptadiene, octadiene or nonadiene.
 9. The lipid membraneaccording to claim 7 wherein the unsaturated C₇-C₉ hydrocarbon is1,6-heptadiene, 1,7-octadiene or 1,8-nonadiene.
 10. The lipid membraneaccording to claim 7, wherein the unsaturated C₇-C₉ hydrocarbon isheptadiene, octadiene, or nonadiene.
 11. The lipid membrane according toclaim 7, wherein the unsaturated C₇-C₉ hydrocarbon is 1,6-heptadiene,1,7-octadiene, or 1,8-nonadiene.
 12. The lipid membrane according toclaim 1, wherein the unsaturated C₇-C₉ hydrocarbon is heptadiene,octadiene or nonadiene.
 13. The lipid membrane according to claim 1,wherein the unsaturated C₇-C₉ hydrocarbon is 1,6-heptadiene,1,7-octadiene or 1,8-nonadiene.
 14. A method for measuring membranepermeability, which comprises the step of measuring the membranepermeability of a substance using the lipid membrane according to anyone of claims 1 to
 13. 15. A screening method, which comprises the stepsof: measuring the membrane permeability of a substance using the lipidmembrane according to any one of claims 1 to 13; and selecting thesubstance if it has the desired membrane permeability.
 16. A kit formeasuring membrane permeability, which comprises an unsaturated C₇-C₉hydrocarbon and a lipid.
 17. A screening kit comprising an unsaturatedC₇-C₉ hydrocarbon, a lipid and an instruction manual for use.